岡山医学会 Acta Medica Okayama 0030-1558 138 1 2026 日本解剖学会第79回中国・四国支部学術集会 28 28 EN Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University No potential conflict of interest relevant to this article was reported.

MDPI AG Acta Medica Okayama 1422-0067 27 5 2026 Fgf10 Gene Dosage from a Single Allele Is Insufficient for Forming Multilayered Epithelial Cells in the Murine Lacrimal Gland 2113 EN Shiori Ikeda Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yuki Tajika Department of Radiological Technology, Gumma Prefectural College of Health Sciences Hirofumi Fujita Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Tetsuya Bando Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Tsutomu Nohno Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Satoru Miyaishi Department of Legal Medicine, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Mutations in the fibroblast growth factor 10 (FGF10) gene in humans cause aplasia of the lacrimal and salivary glands (ALSG). In patients with ALSG, heterozygous loss-of-function mutations are found, and FGF10 haploinsufficiency results in the absence of these secretory organs. Lacrimal glands (LGs) are formed through epithelial thickening, budding, and branching morphogenesis. To compare the variable phenotypes of the Fgf10+/− Harderian glands (HGs) previously reported, we examined the development of LGs in wild-type (WT), Fgf10+/−, and Fgf10-null mice. Pax6 immunostaining was performed to visualize the LG primordia from embryonic day 15.5 (E15.5) onwards. In situ hybridization of the genes encoding the epithelial receptor of FGF10, FGFR2b, and its other ligands was performed to determine their potential involvement in LG development. LG primordia were not observed in Fgf10+/− mice bilaterally at E16.5 or later stages. At E15.5, budding from the developing conjunctival epithelium (CE) was observed in a small fraction of the Fgf10+/− LG primordia. In contrast, the Fgf10-null CE failed to promote budding. Among Fgf1, Fgf3, Fgf7, Fgf10, and Fgf22, Fgf10 was expressed in the mesenchyme surrounding developing LG epithelial cells, whereas Fgf1 was expressed in the LG epithelium of WT mice. Fgf7 was initially expressed in the mesenchyme surrounding the nascent LG epithelium, but its expression subsequently became diffused. Thus, we conclude that among the FGFR2b ligands, initial LG formation is dependent on the mesenchymal factors FGF10 and FGF7, and FGF1 is likely to function as an epithelial factor in the LG primordia. A single allele of Fgf10 was found to be insufficient to support the budding process during LG morphogenesis. No potential conflict of interest relevant to this article was reported.

The Company of Biologists Acta Medica Okayama 0950-1991 152 22 2025 ROS produced by Dual oxidase regulate cell proliferation and haemocyte migration during leg regeneration in the cricket dev204763 EN Misa Okumura-Hirono Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshimasa Hamada Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Motoo Araki Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Many animals regenerate lost body parts through several signalling pathways; however, the triggers that initiate regeneration remain unclear. In the present study, we focused on the role of reactive oxygen species (ROS) produced by the NADPH oxidase Dual oxidase (Duox) during cricket leg regeneration. The results showed that ROS levels were upregulated during leg regeneration and decreased by DuoxRNAi. In DuoxRNAi nymphs, wound closure and scab formation were incomplete 2 days after amputation, and hypertrophy occurred in the distal region of the regenerating legs at 5 days after amputation. In addition, the hypertrophic phenotype was induced by DuoxARNAi and NADPH oxidase inhibitor treatment. During hypertrophy, haemocytes, including plasmatocytes, oenocytoids and granulocytes, accumulated. Proliferation of haemocytes in regenerating legs was not increased by DuoxRNAi; however, haemocyte accumulation was regulated by the Spatzle (Spz) family molecules, which are Toll receptor ligands. As the exoskeleton of DuoxRNAi nymphs was thinner than that of the control, excessive haemocyte accumulation can cause hypertrophy in DuoxRNAi nymphs. Thus, Duox-derived ROS are involved in wound healing and haemocyte accumulation through the Spz/Toll signalling pathway during leg regeneration in crickets. No potential conflict of interest relevant to this article was reported.

American Chemical Society (ACS) Acta Medica Okayama 0002-7863 2025 Optogenetic Cancer Therapy Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin-3 (AR3) EN Shin Nakao Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keita Sato Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Naoya Kemmotsu Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Togashi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuki Sudo Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Medicines used for cancer treatment often cause serious side effects by damaging normal cells due to nonspecific diffusion. To address this issue, we previously developed an optical method to induce apoptotic cell death via intracellular pH alkalinization using the outward proton pump rhodopsin, Archaerhodopsin-3 (AR3) in various noncancer model cells in vitro and in vivo. In this study, we applied this method to cancer cells and tumors to evaluate its potential as an anticancer therapeutic strategy. First, we confirmed that AR3-expressing murine cancer cell lines (MC38, B16F10) showed apoptotic cell death upon green light irradiation, as indicated by increased levels of cell death and apoptosis-related markers. Next, we established stable AR3-expressing MC38 and B16F10 cells by using viral vectors. When these AR3-expressing cells were subcutaneously transplanted into C57BL/6 mice, the resulting tumors initially grew at a rate comparable to that of control tumors lacking AR3 expression or light stimulation. However, upon green light irradiation, AR3-expressing tumors exhibited either a marked reduction in size or significantly suppressed growth, accompanied by the induction of apoptosis signals and decreased proliferation signals. These results demonstrate that AR3-mediated cell death has potent antitumor effects both in vitro and in vivo. This optical method thus holds promise as a novel cancer therapy with potentially reduced side effects. No potential conflict of interest relevant to this article was reported.

Oxford University Press (OUP) Acta Medica Okayama 1365-2672 136 10 2025 Gut dysbiosis allows foodborne salmonella colonization in edible crickets: a probiotic strategy for enhanced food safety lxaf217 EN Shuma Tsuji Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Osamu Matsushita Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jumpei Uchiyama Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kenji Yokota Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuyoshi Gotoh Department of Medical Laboratory Science, Okayama University Graduate School of Health Sciences Aims: Edible insects, including crickets, represent a promising protein source, yet concerns over foodborne pathogens limit consumer acceptance. This study investigated whether gut microbiota modulates colonization by Salmonella enterica subsp. enterica serovar Enteritidis (SE) in the two-spotted cricket (Gryllus bimaculatus).<br> Methods and Results: Under standard conditions, SE was undetectable in crickets despite prolonged exposure; however, antibiotic-induced dysbiosis enabled stable SE colonization. Long-read 16S rRNA sequencing revealed significant microbiota shifts, notably a reduction in Lactococcus garvieae. In vitro assays showed strong inhibitory effects of L. garvieae against SE, and supplementation of dysbiotic crickets with L. garvieae reduced SE colonization by ∼1000-fold.<br> Conclusions: The native cricket gut microbiota, especially L. garvieae, plays a protective role against SE colonization. Enhancing beneficial gut bacteria could mitigate pathogen risks and promote edible insects as a sustainable protein. No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0021-9258 301 7 2025 A repertoire of visible light–sensitive opsins in the deep-sea hydrothermal vent shrimp Rimicaris hybisae 110291 EN Yuya Nagata Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Norio Miyamoto Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Keita Sato Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yosuke Nishimura Research Center for Bioscience and Nanoscience (CeBN), Research Institute for Marine Resources Utilization, Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Yuki Tanioka School of Pharmaceutical Sciences, Okayama University Yuji Yamanaka School of Pharmaceutical Sciences, Okayama University Susumu Yoshizawa Atmosphere and Ocean Research Institute, The University of Tokyo Kuto Takahashi Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kohei Obayashi Department of Biology, Graduate School of Science, Kobe University Hisao Tsukamoto Department of Biology, Graduate School of Science, Kobe University Ken Takai Institute for Extra-Cutting-Edge Science and Technology Avant-Garde Research (X-Star), Japan Agency for Marine-Earth Science and Technology (JAMSTEC) Hideyo Ohuchi Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takahiro Yamashita Department of Biophysics, Graduate School of Science, Kyoto University Yuki Sudo Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Keiichi Kojima Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Unlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457–517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light–sensitive opsins, including the uniquely tuned Opn5. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1472-6831 24 1 2024 Histological differences related to autophagy in the minor salivary gland between primary and secondary types of Sjögren's syndrome 1099 EN Hitomi Ono-Minagi Department of Cytology and Histology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Tsutomu Nohno Department of Cytology and Histology, Okayama University Medical School Kiyofumi Takabatake Department of Oral Pathology and Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takehiro Tanaka Department of Pathology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takayuki Katsuyama Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Hospital Kohta Miyawaki Division of Precision Medicine, Kyushu University School of Medicine Jun Wada Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Soichiro Ibaragi Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Seiji Iida Department of Oral and Maxillofacial Reconstructive Surgery, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tadashi Yoshino Department of Pathology, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine Hitoshi Nagatsuka Department of Oral Pathology and Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takayoshi Sakai Department of Rehabilitation for Orofacial Disorders, Osaka University Graduate School of Dentistry Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Some forms of Sjögren’s syndrome (SS) follow a clinical course accompanied by systemic symptoms caused by lymphocyte infiltration and proliferation in the liver, kidneys, and other organs. To better understand the clinical outcomes of SS, here we used minor salivary gland tissues from patients and examine their molecular, biological, and pathological characteristics. A retrospective study was performed, combining clinical data and formalin-fixed paraffin-embedded (FFPE) samples from female patients over 60 years of age who underwent biopsies at Okayama University Hospital. We employed direct digital RNA counting with nCounter® and multiplex immunofluorescence analysis with a PhenoCycler™ on the labial gland biopsies. We compared FFPE samples from SS patients who presented with other connective tissue diseases (secondary SS) with those from stable SS patients with symptoms restricted to the exocrine glands (primary SS). Secondary SS tissues showed enhanced epithelial damage and lymphocytic infiltration accompanied by elevated expression of autophagy marker genes in the immune cells of the labial glands. The close intercellular distance between helper T cells and B cells positive for autophagy-associated molecules suggests accelerated autophagy in these lymphocytes and potential B cell activation by helper T cells. These findings indicate that examination of FFPE samples from labial gland biopsies can be an effective tool for evaluating molecular histological differences between secondary and primary SS through multiplexed analysis of gene expression and tissue imaging. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2221-3759 12 2 2024 Harderian Gland Development and Degeneration in the Fgf10-Deficient Heterozygous Mouse 16 EN Shiori Ikeda Department of Cytology and Histology, Medical School, Okayama University Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hirofumi Fujita Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hitomi Ono-Minagi Department of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Satoru Miyaishi Department of Legal Medicine, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Tsutomu Nohno Department of Cytology and Histology, Medical School, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The mouse Harderian gland (HG) is a secretory gland that covers the posterior portion of the eyeball, opening at the base of the nictitating membrane. The HG serves to protect the eye surface from infection with its secretions. Mice open their eyelids at about 2 weeks of age, and the development of the HG primordium mechanically opens the eye by pushing the eyeball from its rear. Therefore, when HG formation is disturbed, the eye exhibits enophthalmos (the slit-eye phenotype), and a line of Fgf10(+/-) heterozygous loss-of-function mice exhibits slit-eye due to the HG atrophy. However, it has not been clarified how and when HGs degenerate and atrophy in Fgf10(+/-) mice. In this study, we observed the HGs in embryonic (E13.5 to E19), postnatal (P0.5 to P18) and 74-week-old Fgf10(+/-) mice. We found that more than half of the Fgf10(+/-) mice had markedly degenerated HGs, often unilaterally. The degenerated HG tissue had a melanized appearance and was replaced by connective tissue, which was observed by P10. The development of HGs was delayed or disrupted in the similar proportion of Fgf10(+/-) embryos, as revealed via histology and the loss of HG-marker expression. In situ hybridization showed Fgf10 expression was observed in the Harderian mesenchyme in wild-type as well as in the HG-lacking heterozygote at E19. These results show that the Fgf10 haploinsufficiency causes delayed or defective HG development, often unilaterally from the unexpectedly early neonatal period. No potential conflict of interest relevant to this article was reported.

Springer Science and Business Media LLC Acta Medica Okayama 0302-766X 391 2 2022 The medaka mutant deficient in eyes shut homolog exhibits opsin transport defects and enhanced autophagy in retinal photoreceptors 249 267 EN Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yang Liu Department of Cytology and Histology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takahiro Yamashita Department of Biophysics, Graduate School of Science, Kyoto University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Eyes shut homolog (EYS) encodes a proteoglycan and the human mutation causes retinitis pigmentosa type 25 (RP25) with progressive retinal degeneration. RP25 most frequently affects autosomal recessive RP patients with many ethnic backgrounds. Although studies using RP models have facilitated the development of therapeutic medications, Eys has been lost in rodent model animals. Here we examined the roles for Eys in the maintenance of photoreceptor structure and function by generating eys-null medaka fish using the CRISPR-Cas9 system. Medaka EYS protein was present near the connecting cilium of wild-type photoreceptors, while it was absent from the eys−/− retina. The mutant larvae exhibited a reduced visual motor response compared with wild-type. In contrast to reported eys-deficient zebrafish at the similar stage, no retinal cell death was detected in the 8-month post-hatching (8-mph) medaka eys mutant. Immunohistochemistry showed a significant reduction in the length of cone outer segments (OSs), retention of OS proteins in the inner segments of photoreceptors, and abnormal filamentous actin network at the base of cone OSs in the mutant retina by 8 mph. Electron microscopy revealed aberrant structure of calyceal processes, numerous vesiculation and lamellar interruptions, and autophagosomes in the eys-mutant cone photoreceptors. In situ hybridization showed an autophagy component gene, gabarap, was ectopically expressed in the eys-null retina. These results suggest eys is required for regeneration of OS, especially of cone photoreceptors, and transport of OS proteins by regulating actin filaments. Enhanced autophagy may delay the progression of retinal degeneration when lacking EYS in the medaka retina. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 1083-351X 299 8 2023 Mammalian type opsin 5 preferentially activates G14 in Gq-type G proteins triggering intracellular calcium response 105020 EN Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Takahiro Yamashita Department of Biophysics, Graduate School of Science, Kyoto University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and G alpha-KO cell line. Beyond the commonly studied major G alpha classes, G alpha q, G alpha 11, G alpha 14, and G alpha 15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type G alpha deletion and rescued by cotransfection with mouse and medaka Gq-type G alpha proteins. Opn5m preferentially activated G alpha 14 and close relatives. Mutational analysis implicated specific regions, including alpha 3-beta 5 and alpha G-alpha 4 loops, alpha G and alpha 4 helices, and the extreme C terminus, in the preferential activation of G alpha 14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and G alpha 14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of G alpha 14 by Opn5m is relevant for UV sensing in specific cell types. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0022-2836 436 5 2024 Molecular Property, Manipulation, and Potential Use of Opn5 and Its Homologs 168319 EN Keita Sato Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Animal opsin is a G-protein coupled receptor (GPCR) and binds retinal as a chromophore to form a photopigment. The Opsin 5 (Opn5) group within the animal opsin family comprises a diverse array of related proteins, such as Opn5m, a protein conserved across all vertebrate lineages including mammals, and other members like Opn5L1 and Opn5L2 found in non-mammalian vertebrate genomes, and Opn6 found in non-therian vertebrate genomes, along with Opn5 homologs present in invertebrates. Although these proteins collectively constitute a single clade within the molecular phylogenetic tree of animal opsins, they exhibit markedly distinct molecular characteristics in areas such as retinal binding properties, photoreaction, and G-protein coupling specificity. Based on their molecular features, they are believed to play a significant role in physiological functions. However, our understanding of their precise physiological functions and molecular characteristics is still developing and only partially realized. Furthermore, their unique molecular characteristics of Opn5-related proteins suggest a high potential for their use as optogenetic tools through more specialized manipulations. This review intends to encapsulate our current understanding of Opn5, discuss potential manipulations of its molecular attributes, and delve into its prospective utility in the burgeoning field of animal opsin optogenetics. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1661-6596 24 18 2023 The Germinal Origin of Salivary and Lacrimal Glands and the Contributions of Neural Crest Cell-Derived Epithelium to Tissue Regeneration 13692 EN Hitomi Ono-Minagi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tsutomu Nohno Department of Cytology and Histology, Okayama University Medical School Takashi Serizawa Department of Physiology, Keio University School of Medicine Yu Usami Department of Oral and Maxillofacial Pathology, Osaka University Graduate School of Dentistry Takayoshi Sakai Department of Rehabilitation for Orofacial Disorders, Osaka University Graduate School of Dentistry Hideyuki Okano Department of Physiology, Keio University School of Medicine Hideyo Ohuchi Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences The vertebrate body comprises four distinct cell populations: cells derived from (1) ectoderm, (2) mesoderm, (3) endoderm, and (4) neural crest cells, often referred to as the fourth germ layer. Neural crest cells arise when the neural plate edges fuse to form a neural tube, which eventually develops into the brain and spinal cord. To date, the embryonic origin of exocrine glands located in the head and neck remains under debate. In this study, transgenic TRiCK mice were used to investigate the germinal origin of the salivary and lacrimal glands. TRiCK mice express fluorescent proteins under the regulatory control of Sox1, T/Brachyury, and Sox17 gene expressions. These genes are representative marker genes for neuroectoderm (Sox1), mesoderm (T), and endoderm (Sox17). Using this approach, the cellular lineages of the salivary and lacrimal glands were examined. We demonstrate that the salivary and lacrimal glands contain cells derived from all three germ layers. Notably, a subset of Sox1-driven fluorescent cells differentiated into epithelial cells, implying their neural crest origin. Also, these Sox1-driven fluorescent cells expressed high levels of stem cell markers. These cells were particularly pronounced in duct ligation and wound damage models, suggesting the involvement of neural crest-derived epithelial cells in regenerative processes following tissue injury. This study provides compelling evidence clarifying the germinal origin of exocrine glands and the contribution of neural crest-derived cells within the glandular epithelium to the regenerative response following tissue damage. No potential conflict of interest relevant to this article was reported.

BMC Acta Medica Okayama 1472-6831 23 1 2023 Evaluation of oral care using MA-T gel for high-risk patients: a pilot study 108 EN Hitomi Ono-Minagi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Nao Gojo Department of Oral‑Facial Disorders, Osaka University Graduate School of Dentistry Tsutomu Nohno Department of Cytology and Histology, Okayama University Medical School Tsuyoshi Inoue Institute for Open and Transdisciplinary Research Initiatives, Osaka University Hideyo Ohuchi Department of Cytology and Histology, Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Takayoshi Sakai Department of Oral‑Facial Disorders, Osaka University Graduate School of Dentistry Background Oral care with gel is a common method for preventing aspiration in high-risk patients. An oral care gel is used to clean and moisturize the oral cavity. However, the effects of gel care on the oral bacteria remain unclear. In this pilot study, we described a matching transformation system (MA-T) for elderly high-risk patients. MA-T is an on-demand aqueous chlorine dioxide solution that provides excellent safety and has various antimicrobial activities, even in the presence of abundant organic compounds. This study investigated the effects of MA-T gel in patients requiring nursing care.<br> Materials and methods Patients who were hospitalized for nursing care were included in this study. No drugs and foods were administered orally. Oral bacteria and intraoral humidity were examined by daily care using MA-T gel. Moreover, oral membranous substances were analyzed and material from the oral cavity was cultured on selective media for identifying opportunistic organisms.<br> Results Membranous substances were present in the oral cavities of all patients. The number of bacteria decreased, and oral moisture improved, after treatment with MA-T gel. Moreover, oral humidity was also controlled with the continued use of MA-T gel. MA-T gels should be used not only for professional care but also on a daily basis for better oral care. Furthermore, the results of bacterial cultures showed that MA-T controls the propagation of opportunistic bacterial infections.<br> Conclusion Membranous substances may be observed in the oral cavity of individuals requiring nursing care for tube feeding. The results of this pilot study suggest that MA-T, a novel disinfectant, can be used for oral care in the elderly to reduce the risk of aspiration-pneumonia. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 17 11 2022 Cysteinyl leukotriene receptor 1 is dispensable for osteoclast differentiation and bone resorption e0277307 EN Hirofumi Fujita Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Aoi Ando Faculty of Medicine, Okayama University Medical School Yohei Mizusawa Faculty of Medicine, Okayama University Medical School Mitsuaki Ono Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Takako Hattori Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Munenori Habuta Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Toshitaka Oohashi Department of Molecular Biology and Biochemistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Satoshi Kubota Department of Biochemistry and Molecular Dentistry, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Cysteinyl leukotriene receptor 1 (CysLTR1) is a G protein-coupled receptor for the inflammatory lipid mediators cysteinyl leukotrienes, which are involved in smooth muscle constriction, vascular permeability, and macrophage chemokine release. The Cysltr1 gene encoding CysLTR1 is expressed in the macrophage lineage, including osteoclasts, and the CysLTR1 antagonist Montelukast has been shown to suppress the formation of osteoclasts. However, it currently remains unclear whether CysLTR1 is involved in osteoclast differentiation and bone loss. Therefore, to clarify the role of CysLTR1 in osteoclastogenesis and pathological bone loss, we herein generated CysLTR1 loss-of-function mutant mice by disrupting the cysltr1 gene using the CRISPR-Cas9 system. These mutant mice had a frameshift mutation resulting in a premature stop codon (Cysltr1 KO) or an in-frame mutation causing the deletion of the first extracellular loop (Cysltr1(Delta 105)). Bone marrow macrophages (BMM) from these mutant mice lost the intracellular flux of calcium in response to leukotriene D-4, indicating that these mutants completely lost the activity of CysLTR1 without triggering genetic compensation. However, disruption of the Cysltr1 gene did not suppress the formation of osteoclasts from BMM in vitro. We also demonstrated that the CysLTR1 antagonist Montelukast suppressed the formation of osteoclasts without functional CysLTR1. On the other hand, disruption of the Cysltr1 gene partially suppressed the formation of osteoclasts stimulated by leukotriene D-4 and did not inhibit that by glutathione, functioning as a substrate in the synthesis of cysteinyl leukotrienes. Disruption of the Cysltr1 gene did not affect ovariectomy-induced osteoporosis or lipopolysaccharide-induced bone resorption. Collectively, these results suggest that the CysLT-CysLTR1 axis is dispensable for osteoclast differentiation in vitro and pathological bone loss, while the leukotriene D-4-CysTR1 axis is sufficient to stimulate osteoclast formation. We concluded that the effects of glutathione and Montelukast on osteoclast formation were independent of CysLTR1. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2221-3759 10 4 2022 Involvement of a Basic Helix-Loop-Helix Gene BHLHE40 in Specification of Chicken Retinal Pigment Epithelium 45 EN Toshiki Kinuhata Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Taro Mito Bio-Innovation Research Center, Tokushima University Satoru Miyaishi Department of Legal Medicine, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences Tsutomu Nohno Department of Cytology and Histology, Okayama University Medical School Hideyo Ohuchi Department of Cytology and Histology, Okayama University Faculty of Medicine, Dentistry and Pharmaceutical Sciences The first event of differentiation and morphogenesis in the optic vesicle (OV) is specification of the neural retina (NR) and retinal pigment epithelium (RPE), separating the inner and outer layers of the optic cup, respectively. Here, we focus on a basic helix-loop-helix gene, BHLHE40, which has been shown to be expressed by the developing RPE in mice and zebrafish. Firstly, we examined the expression pattern of BHLHE40 in the developing chicken eye primordia by in situ hybridization. Secondly, BHLHE40 overexpression was performed with in ovo electroporation and its effects on optic cup morphology and expression of NR and RPE marker genes were examined. Thirdly, we examined the expression pattern of BHLHE40 in LHX1-overexpressed optic cup. BHLHE40 expression emerged in a subset of cells of the OV at Hamburger and Hamilton stage 14 and became confined to the outer layer of the OV and the ciliary marginal zone of the retina by stage 17. BHLHE40 overexpression in the prospective NR resulted in ectopic induction of OTX2 and repression of VSX2. Conversely, BHLHE40 was repressed in the second NR after LHX1 overexpression. These results suggest that emergence of BHLHE40 expression in the OV is involved in initial RPE specification and that BHLHE40 plays a role in separation of the early OV domains by maintaining OTX2 expression and antagonizing an NR developmental program. No potential conflict of interest relevant to this article was reported.

The Company of Biologists Acta Medica Okayama 0950-1991 149 8 2021 Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages dev199916 EN Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Misa Okumura Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yuki Bando Faculty of Medicine, Okayama University Medical School Marou Hagiwara Faculty of Medicine, Okayama University Medical School Yoshimasa Hamada Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yoshiyasu Ishimaru Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University Taro Mito Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University Eri Kawaguchi Division of Biological Science, Graduate School of Science, Kyoto University Takeshi Inoue Division of Biological Science, Graduate School of Science, Kyoto University Kiyokazu Agata Division of Biological Science, Graduate School of Science, Kyoto University Sumihare Noji Division of Bioscience and Bioindustry, Graduate School of Technology, Industrial and Social Sciences, Tokushima University Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4425 13 2 2022 Dkk3/REIC Deficiency Impairs Spermiation, Sperm Fibrous Sheath Integrity and the Sperm Motility of Mice 285 EN Ruizhi Xue Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Wenfeng Lin Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Jingkai Sun Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Rie Kinoshita Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Kazuhiko Ochiai Laboratory of Veterinary Hygiene, Nippon Veterinary and Life Science University Junichiro Futami Department of Interdisciplinary Science and Engineering in Health Systems, Okayama University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Masakiyo Sakaguchi Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Zhengyan Tang Department of Urology, Xiangya Hospital, Central South University Peng Huang Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Yasutomo Nasu Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University The role of Dickkopf-3 (Dkk3)/REIC (The Reduced Expression in Immortalized Cells), a Wnt-signaling inhibitor, in male reproductive physiology remains unknown thus far. To explore the functional details of Dkk3/REIC in the male reproductive process, we studied the Dkk3/REIC knock-out (KO) mouse model. By examining testicular sections and investigating the sperm characteristics (count, vitality and motility) and ultrastructure, we compared the reproductive features between Dkk3/REIC-KO and wild-type (WT) male mice. To further explore the underlying molecular mechanism, we performed RNA sequencing (RNA-seq) analysis of testicular tissues. Our results showed that spermiation failure existed in seminiferous tubules of Dkk3/REIC-KO mice, and sperm from Dkk3/REIC-KO mice exhibited inferior motility (44.09 +/- 8.12% vs. 23.26 +/- 10.02%, p < 0.01). The Ultrastructure examination revealed defects in the sperm fibrous sheath of KO mice. Although the average count of Dkk3/REIC-KO epididymal sperm was less than that of the wild-types (9.30 +/- 0.69 vs. 8.27 +/- 0.87, x10(6)), neither the gap (p > 0.05) nor the difference in the sperm vitality rate (72.83 +/- 1.55% vs. 72.50 +/- 0.71%, p > 0.05) were statistically significant. The RNA-seq and GO (Gene Oncology) enrichment results indicated that the differential genes were significantly enriched in the GO terms of cytoskeleton function, cAMP signaling and calcium ion binding. Collectively, our research demonstrates that Dkk3/REIC is involved in the process of spermiation, fibrous sheath integrity maintenance and sperm motility of mice. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0021-9967 529 2021 The Opsin 3/Teleost multiple tissue opsin system: mRNA localization in the retina and brain of medaka (Oryzias latipes) 2484 2516 EN Keita Sato Department of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Khine Nwe Nwe Department of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University The photoreceptor protein, opsin, is one of the major components for vision and photoreceptive function in animals. Although many opsins have been discovered from animal genomes, only a few nonimage‐forming functions mediated by opsins have been identified. Understanding the mRNA distribution of photoreceptor proteins is one crucial step in uncovering their photoreceptive function in animals. Here, we focus on the medaka fish (Oryzias latipes) Opsin 3 (Opn3)/Teleost multiple opsin (Tmt) system, which constitutes a separate phylogenetic group, having putative blue light photoreceptors for nonimage‐forming functions. In medaka, there is one opn3 and five tmt‐opsin orthologs. The expression pattern of the opn3/tmt‐opsins in the retina and brain was investigated by in situ hybridization. mRNAs for opn3/tmt‐opsins were distributed in the retinal ganglion cells as well as interneurons and specific brain nuclei. Specifically, hybridization signals were observed in the glutamate decarboxylase 1 (gad1)‐expressing amacrine cells for opn3, tmt1a, tmt1b, and tmt2, in the caudal lobe of the cerebellum for tmt1b and tmt2, in the cranial nerve nuclei for opn3, tmt1a, tmt1b, tmt2, and in the rostral pars distalis (adenohypophysis) for opn3. These expression patterns suggest that blue light sensing in the fish retina and brain may be involved in the integration of visual inputs, vestibular function, somatosensation, motor outputs, and pituitary endocrine regulation. No potential conflict of interest relevant to this article was reported.

Public Library of Science Acta Medica Okayama 1932-6203 15 10 2020 Fgf10-CRISPR mosaic mutants demonstrate the gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung e0240333 EN Munenori Habuta Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Akihiro Yasue Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Ken-Ichi T. Suzuki Department of Mathematical and Life Sciences, Hiroshima University Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hitomi Kono Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Ayuko Takayama Center for the Development of New Model Organisms, National Institute for Basic Biology Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Satoru Miyaishi Department of Legal Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute of Advanced Medical Sciences, Tokushima University Eiji Tanaka Department of Orthodontics and Dentofacial Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences CRISPR/Cas9-mediated gene editing often generates founder generation (F0) mice that exhibit somatic mosaicism in the targeted gene(s). It has been known thatFibroblast growth factor 10(Fgf10)-null mice exhibit limbless and lungless phenotypes, while intermediate limb phenotypes (variable defective limbs) are observed in theFgf10-CRISPR F0 mice. However, how the lung phenotype in theFgf10-mosaic mutants is related to the limb phenotype and genotype has not been investigated. In this study, we examined variable lung phenotypes in theFgf10-targeted F0 mice to determine if the lung phenotype was correlated with percentage of functionalFgf10genotypes. Firstly, according to a previous report,Fgf10-CRISPR F0 embryos on embryonic day 16.5 (E16.5) were classified into three types: type I, no limb; type II, limb defect; and type III, normal limbs. Cartilage and bone staining showed that limb truncations were observed in the girdle, (type I), stylopodial, or zeugopodial region (type II). Deep sequencing of theFgf10-mutant genomes revealed that the mean proportion of codons that encode putative functional FGF10 was 8.3 +/- 6.2% in type I, 25.3 +/- 2.7% in type II, and 54.3 +/- 9.5% in type III (mean +/- standard error of the mean) mutants at E16.5. Histological studies showed that almost all lung lobes were absent in type I embryos. The accessory lung lobe was often absent in type II embryos with other lobes dysplastic. All lung lobes formed in type III embryos. The number of terminal tubules was significantly lower in type I and II embryos, but unchanged in type III embryos. To identify alveolar type 2 epithelial (AECII) cells, known to be reduced in theFgf10-heterozygous mutant, immunostaining using anti-surfactant protein C (SPC) antibody was performed: In the E18.5 lungs, the number of AECII was correlated to the percentage of functionalFgf10genotypes. These data suggest theFgf10gene dose-related loss of the accessory lobe and decrease in the number of alveolar type 2 epithelial cells in mouse lung. Since dysfunction of AECII cells has been implicated in the pathogenesis of parenchymal lung diseases, theFgf10-CRISPR F0 mouse would present an ideal experimental system to explore it. No potential conflict of interest relevant to this article was reported.

Association for Research in Vision and Ophthalmology Acta Medica Okayama 0146-0404 61 5 2020 KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells 38 EN Yuki Kanzaki Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hirofumi Fujita Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Keita Sato Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Mio Hosokawa Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hiroshi Matsumae Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Fumio Shiraga Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Yuki Morizane Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Hideyo Ohuchi Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. </br> Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. </br> Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. </br> Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16. No potential conflict of interest relevant to this article was reported.

Okayama University Medical School Acta Medica Okayama 0386-300X 74 3 2020 Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland 199 208 EN Hirofumi Fujita Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Tetsuya Bando Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Seiichi Oyadomari Division of Molecular Biology, Institute for Genome Research, University of Tokushima Kazuhiko Ochiai Department of Basic Science, School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University Masami Watanabe Department of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Hiromi Kumon Innovation Center Okayama for Nanobio-Targeted Therapy, Okayama University Hideyo Ohuchi Department of Cytology and Histology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences Original Article 10.18926/AMO/59950 Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland. No potential conflict of interest relevant to this article was reported.

Company of Biologists Acta Medica Okayama 0950-1991 142 17 2015 Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus 2916 2927 EN Yoshimasa Hamada Tetsuya Bando Taro Nakamura Yoshiyasu Ishimaru Taro Mito Sumihare Noji Kenji Tomioka Hideyo Ohuchi Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)RNAi and was increased by Gb'UtxRNAi. Regenerated Gb'E(z)RNAi cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'UtxRNAi cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)RNAi regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'UtxRNAi regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression. No potential conflict of interest relevant to this article was reported.

岡山医学会 Acta Medica Okayama 0030-1558 125 3 2013 組織形成因子Fgf10 229 234 EN Hideyo Ohuchi No potential conflict of interest relevant to this article was reported.